Phosphatidylethanol in post-mortem blood: A comparative study of blood matrices and its stability at routine storage conditions

Abstract

Determination of alcohol markers in post-mortem cases can be useful to classify drinking habits and potential alcohol habituation prior to death. Phosphatidylethanol (PEth) is a direct alcohol marker and is already commonly used in a variety of contexts. However, its use in the field of post-mortem toxicology has been scarcely investigated so far. To evaluate its validity, PEth was determined in routinely collected post-mortem heart blood and femoral vein blood. The stability of PEth under routine storage conditions (-20°C) for a period of 60 days was examined. Post-mortem blood was collected during medicolegal autopsies and aliquoted. Parts of the samples were used to create dried blood spots (DBS) directly after collection. Further DBS were created using aliquots stored at -20°C on days 1, 2, 7, 14, 30, and 60. LC-MS/MS was used for quantitative PEth analysis, and initial blood alcohol was determined using GC-FID. Blood was collected from 50 different post-mortem cases. The heart blood/femoral blood ratio of PEth concentrations varied from 0.32 to 2.36 (mean = approx. 1.00), indicating a good comparability in total but a strong interindividual variation. In all PEth-positive samples, the PEth concentrations increased by approximately 20% after 24 hours and 70% after 60 days. Post-sampling formation of PEth was also found in blood samples without detectable amounts of ethanol. Neither storage at -80°C nor the addition of sodium metavanadate led to satisfactory stability of PEth. Based on our findings, the (sole) use of PEth for post-mortem toxicology caseworks is not recommended.

Keywords: alcohol biomarker; dried blood spots; femoral vein blood; forensic toxicology; heart blood; phospholipase D.

FIGURE 1

Simplified depiction of sampling handling. Heart blood and femoral vein blood samples were collected during medicolegal autopsies, and 20 μL were applied to Whatman™ filter paper to create dried blood spots (DBS) for day 0 (t = 0). Remaining blood was aliquoted in glass tubes and stored at −20°C. After days (t =) 1, 2, 7, 14, 30, and 60, the blood aliquots were used to create DBS. All DBS were created in duplicate and stored at room temperature in a zip‐lock bag containing desiccant until analysis using LC–MS/MS.

FIGURE 2

Stability of PEth 16:0/18:1 (left column) and PEth 16:0/18:2 (right column) in post‐mortem heart blood (HB). From top to bottom, the graphs depict the mean values and corresponding variances at each time point. (A) For total initial PEth‐positive samples (including samples with and without detectable blood alcohol concentrations (BACs)). (B) For PEth‐positive samples with positive BACs. (C) For PEth‐positive samples with negative BACs. HB samples were applied as dried blood spots after medicolegal autopsy (day 0), and aliquots were then stored at −20°C. Further DBS were created after 1, 2, 7, 14, 30, and 60 days. All samples belonging to one post‐mortem subject were analyzed in one analytical run. PEth values on day 0 were normalized to 100%, and the percentage deviation for the other sampling days is depicted.

FIGURE 3

Stability of PEth 16:0/18:1 (left column) and PEth 16:0/18:2 (right column) in post‐mortem femoral vein blood (FB). From top to bottom, the graphs depict the mean values and corresponding variances at each time point. (A) For total initial PEth‐positive samples (including samples with and without detectable blood alcohol concentrations (BACs)). (B) For PEth‐positive samples with positive BACs. (C) For PEth‐positive samples with negative BACs. FB samples were applied as dried blood spots after medicolegal autopsy (day 0), and aliquots were then stored at −20°C. Further DBS were created after 1, 2, 7, 14, 30, and 60 days. All samples belonging to one post‐mortem subject were analyzed in one analytical run. PEth values on day 0 were normalized to 100%, and the percentage deviation for the other sampling days is depicted.

FIGURE 4

Influence of the storage conditions on the PEth 16:0/18:1 (left) and PEth 16:0/18:2 (right) concentrations (n = 6). The graphs depict the mean values and corresponding variances for each time point. Post‐mortem heart blood (HB) samples were applied as dried blood spots after medicolegal autopsy (day 0), and aliquots were then stored at different storage conditions: at −20°C without addition of NaVO₃ (blue circles), at −80°C without addition of NaVO₃ (orange triangles) and at −20°C with addition of NaVO₃ [0.2 mM] (green squares). Further DBS were created after 7, 14, and 21 days. All samples belonging to one post‐mortem subject were analyzed in one analytical run.

FIGURE 5

Comparability of heart blood (HB) and femoral vein blood (FB) for the PEth homologues 16:0/18:1 (left) and 16:0/18:2 (right). Concentrations of PEth homologues (n = 24) in HB were divided by the concentrations of the corresponding PEth homologue in FB to calculate the HB/FB ratio for each observed time point.

FIGURE 6

Ratio of PEth 16:0/18:1 vs. PEth 16:0/18:2 in heart blood HB (left, n = 27) and femoral vein blood FB (right, n = 25). Concentrations of PEth 16:0/18:1 were divided by the concentrations of PEth 16:0/18:2 in each blood matrix and for each observed time point.